Microbial proteases are of considerable interest for both theoretical and practical reasons. The proteases are often extracellular and can be isolated in active form from a spent culture filtrate.
The proteolytic enzymes are generally divided into four groups on the basis of their pH optima, sensitivity to diisopropylphosphofluoridate (DFP) and thiol reagents, and dependence on metals for activity. Thus, one distinguishes:
1 Acid proteases; PA1 2 DFP-sensitive proteases (serine alkaline proteases); PA1 3 metal chelator-sensitive proteases; PA1 4 thiol proteases.
The first group includes such enzymes as microbial rennins and has a value in dairy industry for milk clotting and cheese ripening. The acid proteases are mostly of fungal origin.
The second group, into which the presently described protease falls, comprises the largest number of known proteases and shows the widest taxonomic distribution. Best characterized are the extracellular subtilisins produced by Bacillus sp. and several other enzymes elaborated by Gram-positive bacteria (e.g., Streptomyces, Arthrobacter) and fungi (e.g., Aspergillus, Saccharomyces). Detergent enzymes are the major industrial use for these proteases. Surprisingly, only a very few Gram-negative bacteria have been found to produce proteases of this category. These are disclosed by Matsubara et al. in P. D. Boyer, Ed. The Enzymes, 3rd Edition, Vol. 3, Pp 721-795, Acad. Press, New York, 1971. Pages 792-794 of this reference are particularly pertinent.
The metal chelator-sensitive proteases form a group of the so-called neutral proteases. These enzymes usually depend on zinc and calcium for activity and stability. Some of the proteases of this group are of use in the beer industry.
The smallest group of proteolytic enzymes of microbial origin is that of thiol proteases. Only a few such proteases have been characterized, e.g., thiol proteases for Streptococcus lactis and Clostridium histolyticum. There is no known industrial application for these enzymes.
The present invention is based on the isolation, characterization and use of an extracellular alkaline serine protease from a soil microorganism, Flavobacterium arborescens. The use of this organism for the production of another enzyme, glucose isomerase, is disclosed and claimed by C. K. Lee in U.S. Pat. No. 4,061,539. Two strains of F. arborescens which are disclosed in this patent as being suitable for the production of glucose isomerase, i.e., NRRL B-11,022 and ATCC 4358, were found to be well suited for the production of protease. The '539 patent does not indicate that an organism of this species is useful in the production of microbial alkaline protease.